Plant vascular tissue-specific expression promoter ProAPL and its application

Abstract

The present invention discloses a plant vascular tissue-specific expression promoter ProAPL and its application, belonging to the technical field of plant genetic engineering. A plant vascular tissue-specific expression promoter ProAPL is provided, and the nucleotide sequence of the vascular tissue-specific expression promoter ProAPL is as shown in SEQ ID NO.1. The present invention provides the poplar vascular tissue-specific expression promoter ProAPL, which can effectively express specifically in the poplar vascular tissue, can specifically exert the function of the exogenous gene expression product in a specific tissue, and regulate the continuous expression of the exogenous gene in the plant vascular tissue.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a plant vascular tissue-specific expression promoter ProAPL and its application. Background Art

[0002] A promoter is a sequence that provides recognition and binding sites for RNA polymerase, and it is located upstream of a gene. Once RNA polymerase locates and binds to the promoter sequence, transcription can be initiated, enabling the orderly progress of the growth, development, and physiological processes of an organism, thus ensuring the normal growth and development of plants. Promoters can be classified into constitutive promoters, tissue-specific promoters, and inducible promoters. Constitutive promoters drive the continuous expression of genes in all cells, tissues, and organs; tissue-specific promoters regulate gene expression only in certain specific parts or organs. To date, the promoters widely used in plant expression vectors are constitutive promoters. For example, the cauliflower mosaic virus (CaMV) 35S promoter and the Agrobacterium tumefaciens nopaline synthase (NOS) promoter are used in the vast majority of dicotyledonous transgenic plants, and the rice OsAct1 promoter and the maize polyubiquitin gene (ZmUbi1) promoter are mainly used in monocotyledonous transgenic plants, and these promoters have proven their effectiveness in inducing plant gene expression. However, since genes driven by constitutive promoters are strongly expressed in different tissues and organs of plants, problems such as slower growth, shorter plants, reduced yields, and even death of transgenic plants compared to control plants have gradually emerged during application. The cambium is a key part of tree growth. It is not only responsible for wood formation but also plays an important role in the radial growth and morphogenesis of trees. By applying a cambium-specific promoter to regulate the expression of genes related to cell division, elongation, and differentiation, the division and proliferation of cambium cells can be promoted, increasing wood yield. The maintenance of cambium cell activity is the guarantee for the continuous thickening of the stems of woody plants. In the research of plant genetic engineering, using a cambium region-specific promoter to drive the specific expression of a target gene in the cambium region of trees can effectively exert the function of the target gene in the cambium region, thereby purposefully improving the quality of trees. Therefore, it is imperative to develop a cambium region-specific promoter for trees for targeted and precise design breeding. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a plant vascular tissue-specific expression promoter ProAPL and its application. The tissue-specific expression promoter provided by the present invention can be effectively and specifically expressed in the vascular tissue of poplar, can specifically exert the function of the expression product of an exogenous gene in a specific tissue, and regulate the continuous expression of the exogenous gene in the plant vascular tissue.

[0004] To achieve the above object, the present invention provides a plant vascular tissue-specific expression promoter ProAPL, and the nucleotide sequence of the vascular tissue-specific expression promoter ProAPL is shown in SEQ ID NO.1.

[0005] Preferably, the vascular tissue-specific expression promoter ProAPL is specifically expressed in the vascular tissue of poplar.

[0006] Preferably, the vascular tissue-specific expression promoter ProAPL is specifically expressed in the cambium of poplar.

[0007] The present invention also provides the application of the plant vascular tissue-specific expression promoter ProAPL in regulating the specific expression of a target gene in the vascular tissue of poplar.

[0008] The present invention also provides the application of the plant vascular tissue-specific expression promoter ProAPL in plant genetic improvement, and the plant is poplar.

[0009] The present invention also provides an expression cassette containing the plant vascular tissue-specific expression promoter ProAPL.

[0010] The present invention also provides a recombinant expression vector containing the plant vascular tissue-specific expression promoter ProAPL.

[0011] The present invention also provides an engineered bacterium containing the plant vascular tissue-specific expression promoter ProAPL.

[0012] The present invention also provides a method for cultivating transgenic plants, including the step of transferring the vascular tissue-specific expression promoter ProAPL, the expression cassette, the recombinant expression vector or the engineered bacterium into a plant; the plant is poplar.

[0013] The present invention also provides the application of the expression cassette, the recombinant expression vector or the engineered bacterium in regulating the specific expression of a target gene in the vascular tissue of poplar.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects:

[0015] The poplar vascular tissue-specific expression promoter ProAPL of the present invention is derived from the genomic DNA of Populus alba×Populus glandulosa‘84K’. The size of the poplar vascular tissue-specific expression promoter ProAPL fragment is 1889 bp and has the following characteristics: (1) It is located at the 5'-end and upstream of the Pop_A10G069473 gene; (2) The base length is 1889 bp; (3) It has the necessary sites for initiating transcription and the transcription start point; (4) It is specifically expressed in the poplar vascular tissue, regulating the continuous expression of foreign genes in specific plant tissues and organs, avoiding energy waste and increasing the effect of transgenes, and reducing problems such as plant dwarfing and gene silencing caused by constitutive expression of genes. It can not only serve for transgenic poplar breeding, but also reserve resources for long-term promoter modification and design. The present invention also discloses a recombinant expression vector containing the tissue-specific expression promoter ProAPL. Under the regulation of the tissue-specific expression promoter ProAPL of the recombinant expression vector, the downstream gene is specifically expressed in the plant vascular tissue, providing a tool for studying the efficient, stable and specific expression of genes in the plant vascular tissue in the field of plant genetic engineering, and having broad application prospects.

[0016] Through the research on the poplar vascular tissue-specific promoter ProAPL, revealing the regulatory mechanism of wood quality formation helps to achieve the directional improvement of wood quality and cultivate new tree varieties that meet the requirements. Using the structural characteristics of the vascular tissue to fuse its tissue-specific promoter with functional genes and develop a large number of special application systems is conducive to the research on developing new genetic tools, increasing the accumulation of vascular bundle biomass and enhancing the stress tolerance of forest trees, and can fully exert the application value of the vascular bundle promoter ProAPL. This will provide a new scientific theoretical basis and practical application value for the molecular mechanism of vascular tissue development and wood formation in woody plants, and provide relevant theoretical and technical support for the molecular variety design of improving the wood properties of fast-growing forest tree species. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 Isolation and identification of the poplar vascular tissue-specific promoter ProAPL, where A is the result of UMAP analysis, B is the expression position of the APL homologous gene pal-pou17833, C is the result of APL AspWood data analysis, and D is the result of APL qPCR;

[0019] Figure 2 Detection results of in-situ expression of PagAPL. Among them, A is the negative control, B is the expression position of PagAPL in the stem. The red marks in the figure represent the hybridization signals of PagAPL, and the scale bar is 200μm;

[0020] Figure 3 Schematic diagram of the vector construction containing the promoter ProAPL;

[0021] Figure 4 For Pro PagAPL ::GUS transgenic plants. Among them, A is the state of leaf dedifferentiation into callus after Agrobacterium transformation, B is the callus induction stage, C is the callus culture, D is the stage of shoot induction, and E is the stage of root induction;

[0022] Figure 5 Transgenic lines of APL. Among them, A is line 1, B is line 2, C is line 3, and D is line 4;

[0023] Figure 6 Measurement results of GUS expression levels in transgenic plants of APL. The red arrows in the figure represent the high-expression lines;

[0024] Figure 7 GUS staining results in transgenic plants of APL. Among them, A is line 1, B is line 2, and C is line 3, and the scale bar is 100μm. Detailed implementation manners

[0025] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range and each smaller range between any other stated value or intermediate value within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0029] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0030] Example 1

[0031] Taking Populus alba×P. glandulosa‘84K’ as the research material, using single-cell RNA-Seq data and wood formation database analysis, to identify Populus vascular tissue-specific genes and their expression sites, clone the corresponding promoters, and verify and evaluate the ability of the specific promoters to drive gene-specific expression.

[0032] I. Research on specific genes in the Populus gene map

[0033] Screen Populus vascular tissue-specific genes according to the Populus gene map: The candidate gene is APL (Pop_A10G069473 in the 84K Populus genome, doi:10.1093 / dnares / dsz020).

[0034] II. Research on the promoter activity of Populus transgenic plants

[0035] (1) Identify the homologous genes of the candidate gene and analyze its tissue expression pattern using qPCR.

[0036] (2) Observe the in-situ expression pattern of the candidate gene through in-situ PCR technology.

[0037] (3) Clone the promoter region (~2000bp) of the APL gene highly expressed in the cambium.

[0038] (4) Construct the promoter sequence obtained in (3) into the pCambia2300 - Promoter - 35S mini - GUS - 35S - eyGFPuv vector and transform Agrobacterium tumefaciens.

[0039] (5) Transform ‘84K’ poplar using the leaf disc method with the vector and obtain transgenic plants for subsequent analysis.

[0040] III. Study on the expression sites of tissue - specific promoters

[0041] (1) Initially identify transgenic plants by observing eyGFPuv and detect the expression level of the GUS gene. Select three lines with relatively high GUS expression multiples for subsequent research.

[0042] (2) Conduct GUS histochemical staining on the selected transgenic poplar plants to explore the location of the expressed gene.

[0043] (3) Verify the function of the promoter APL in specifically driving gene expression.

[0044] IV. Co - expression network analysis of vascular tissue - specific genes

[0045] Use the data from poplar gene mapping studies to detect the expression of APL in different tissues. The data for the heatmap was log2 - transformed.

[0046] V. Fluorescent quantitative PCR analysis of the expression level of target genes

[0047] (1) Harvest different tissues from ‘84K’ plants grown in soil for two months: leaves, phloem, cambium, xylem, and roots. Isolate total RNA using the CTAB method, and reverse - transcribe 1 μg of the quantified total RNA. React using the Power 2×SYBR Real - time PCR Premix (TransGen Biotech, Beijing, China) on a LightCycler® 480 thermal cycler (Roche, Germany). Use UBQ as an internal reference (to quantify the relative transcriptional level of this gene in each sample). Use the 2 -ΔΔCt method to analyze the gene expression level.

[0048] (2) Analyze the expression of the APL gene using real - time fluorescent quantitative PCR.

[0049] VI. In - situ PCR localization of gene expression

[0050] (1) Freshly harvested plant tissues (the 7th internode of the stem of the '84K' tissue-cultured plant) were immediately fixed in formaldehyde solution, and the penetration of the fixative was enhanced by vacuum infiltration. Then, the samples were embedded in agarose, trimmed, and sectioned on a vibratome. 50-μm-thick sections were collected into microtubes. DNase (Takara) treatment, reverse transcription (Takara), and in situ PCR were performed in centrifuge tubes using a standard thermal cycler.

[0051] (2) Genomic DNA was removed by DNase and converted into cDNA by reverse transcription. The cDNA was amplified by standard gene-specific PCR incorporating digoxigenin (DIG)-labeled nucleotides. The sections were incubated with an anti-DIG antibody conjugated to alkaline phosphatase.

[0052] (3) The addition of a specific substrate for alkaline phosphatase allowed for colorimetric detection of the DIG-labeled PCR products and their localization as RNA synthesis sites. Then, the sections were transferred to microscope slides and observed using bright-field microscopy (Leica DM6B).

[0053] VII. Vector construction and creation of transgenic poplars

[0054] (1) An 1889-bp APL promoter (ProAPL, nucleotide sequence shown in SEQ ID NO.1) was cloned from the genomic DNA of Populus alba × Populus glandulosa '84K'.

[0055]

[0056] (2) The Pro PagAPL ::GUS construct was generated using the modified vector pCambia2300 - Promoter - 35S mini - GUS - 35S - eyGFPuv and verified by Sanger sequencing.

[0057] (3) Subsequently, the vector was transformed into Agrobacterium tumefaciens GV3101 using Agrobacterium - mediated transformation. Agrobacterium cells were collected and resuspended to an OD 600 between 0.3 and 0.4. The leaf discs of Populus alba×P. glandulosa tissue - cultured were soaked in the resuspended cells for 20 min, and then the leaf discs were transferred to the medium for inducing callus formation and co - cultured in the dark at room temperature for 2 d. At 25 °C, they were cultured on 40 mg·L -1 Kan medium for 30 d to induce the formation of stems, leaves and roots.

[0058] VIII. Identification of transgenic plants

[0059] (1) Observation of eyGFPuv

[0060] Since the modified vector carries the eyGFPuv tag, after irradiating the transgenic poplar seedlings with a uv lamp, the target plants will emit green fluorescence, and thus the target transgenic plants can be screened.

[0061] (2) Extract the DNA of transgenic plants and detect the expression level of the GUS gene.

[0062] Extract DNA from the APL transgenic plants screened in (1) to detect the GUS expression level, and select 3 lines with higher expression levels for subsequent experiments.

[0063] IX. Histochemical GUS staining and microscopic observation

[0064] (1) Incubate the transgenic plants in 90% acetone - ice for 2 h, wash 3 times with washing buffer, then place them in 1 mM X - Gluc washing buffer and incubate in the dark at 37 °C for 8 h, and wash the plants with 75% ethanol.

[0065] (2) Section with a Leica VT1200S, with a section thickness of 50 μm, and observe under a Leica DM6B microscope. The washing buffer contains: 0.2 mol·L -1 NaH 2 PO 4 , 0.2 mol·L -1 Na 2 HPO 4 , 2 mmol·L -1 K 3 [Fe(CN) 6, 2 mmol·L -1 K 4 [Fe(CN) 6 .

[0066] X. Quantitative Analysis of GUS Activity

[0067] The GUS activity of transgenic plants was detected using a GUS quantitative detection kit (SL7161). GUS reacts with 4-MUG to generate the fluorescent substance 4-MU, which has the highest absorption peak at the excitation wavelengths of 365 nm and 455 nm. Well-ground samples were mixed with 1 mL of extraction buffer and vortexed vigorously for 30 s, then centrifuged at 12,000 rpm for 10 min. Then, 150 μL of the supernatant was added to 150 μL of the 4-MUG substrate solution to form a reaction solution. The mixture was heated at 37 °C, and 100 μL of the reaction solution was taken out every 10 min and 900 μL of the stop solution was added. The GUS activity value in the total protein was finally recorded as 4-MU pmol min -1 ·mg -1 .

[0068] XI. Results

[0069] 1. Identification of Poplar Vascular Tissue-Specific Genes

[0070] Using UMAP analysis (as shown in A in Figure 1 ), it was found that the APL homologous gene PagAPL (Pop_A10G069473 in the 84K poplar genome, doi: 10.1093 / dnares / dsz020) was mainly expressed in sieve tube elements and the cambium region (as shown in B in Figure 1 ), indicating that the gene might be driven by a specific promoter. To further confirm the expression of the gene during wood development, AspWood data analysis showed that this gene was expressed in the cambium near the phloem (as shown in C in Figure 1 ), and the expression pattern was the same as that of the UMAP analysis. qPCR showed that the expression in the stem was higher than that in the leaves and roots, and PagAPL was highly expressed in the cambium (as shown in D in Figure 1 ).

[0071] 2. In Situ PCR

[0072] The in situ expression of PagAPL was explored by in situ PCR technology, and as shown in the results in A in Figure 2 and B in Figure 2 , the PagAPL gene was mainly expressed in the cambium.

[0073] 3. Vector Construction

[0074] To further determine the detailed expression pattern, as shown in Figure 3As shown, the promoter region ProAPL of PagAPL was cloned and homologous recombination was carried out using two restriction enzyme sites, EcoR I and XBa I. The ProAPL promoter was fused with the 35S mini promoter and the β-glucuronidase (GUS) reporter gene for expression.

[0075] 4. Creation of transgenic plants

[0076] As Figure 4 shown in A Figure 4 shown in B Figure 4 shown in C Figure 4 shown in D and Figure 4 shown in E, transgenic plants of Pro PagAPL ::GUS were obtained by transforming Populus alba×P. glandulosa‘84K’ leaves with Agrobacterium tumefaciens. The vector was modified using the artificially enhanced green fluorescent protein (eyGFPuv) to make it visible, making it easier to find transgenic plants for measuring GUS enzyme activity and further exploring the activity characteristics of specific promoters.

[0077] 5. Identification of transgenic plants

[0078] As Figure 5 shown in A Figure 5 shown in B Figure 5 shown in C and Figure 5 shown in D, the vector used carried the eyGFPuv tag, and transgenic plants could emit green fluorescence under the irradiation of a handheld fluorescent lamp, enabling rapid identification of whether they were transgenic plants. Four transgenic lines of the APL gene were obtained, namely Line 1, Line 2, Line 3, and Line 4.

[0079] To further explore the activity characteristics of specific promoters, the GUS expression levels in transgenic plants were detected (as Figure 6 shown), and the results showed that compared with the wild type, the GUS expression levels in transgenic plants were all increased. Three lines with higher GUS expression levels in the gene, namely #1, #2, and #3 in APL, were selected for subsequent experiments.

[0080] 6. GUS histochemical staining

[0081] GUS histochemical staining was performed on transgenic poplar plants to explore its expression location. GUS staining showed that GUS signals were present in the vascular tissue (as Figure 7 shown in A Figure 7 shown in B and Figure 7 shown in C). In the Pro PagAPL ::GUS sections, GUS signals were mainly in the phloem and cambium. This indicates that the promoter ProAPL of the present invention exhibits a vascular tissue-specific expression pattern in poplar and can be applied to initiate the expression of corresponding foreign target genes in genetic engineering.

[0082] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A poplar phloem and cambium specific expression promoter ProAPL, characterized in that: The nucleotide sequence of the poplar phloem and cambium specific expression promoter ProAPL is shown in SEQ ID NO.

1.

2. Use of the poplar phloem and cambium specific expression promoter ProAPL as claimed in claim 1 in regulating the specific expression of a target gene in the poplar phloem and cambium.

3. The use of the poplar phloem and cambium specific expression promoter ProAPL in plant genetic improvement as claimed in claim 1, characterized in that: The plant is poplar.

4. An expression cassette containing the poplar phloem and cambium specific expression promoter ProAPL according to claim 1.

5. A recombinant expression vector containing the poplar phloem and cambium specific expression promoter ProAPL according to claim 1.

6. An engineered bacterium containing the poplar phloem and cambium specific expression promoter ProAPL according to claim 1.

7. A method for cultivating transgenic plants, characterized in that: The method comprises the steps of transferring the poplar phloem and cambium specific expression promoter ProAPL described in claim 1, the expression cassette described in claim 4, the recombinant expression vector described in claim 5 or the engineered bacteria described in claim 6 into a plant; the plant is poplar.

8. Use of the expression cassette according to claim 4, the recombinant expression vector according to claim 5 or the engineered bacteria according to claim 6 in regulating the specific expression of a target gene in the phloem and cambium of poplar.

Images